One of the major deficiencies of current medical countermeasures is that current oximes cannot reactivate nerve agent-inhibited acetylcholinesterase (AChE) that has aged. This proposal focuses on identifying organic molecules that will enable aged nerve agent-AChE to be reactivated. The objective of this proposal is to discover alkylating compounds that selectively react with aged agent-inhibited AChE to form stable methylalkylphosphonate-AChE adducts that can be reactivated with conventional oximes. This drug discovery effort will be conducted as an in vitro research project in which the alkylation process and the oxime reactivation process will be investigated as separate and sequential problems. The specific aims of this proposal are: to design and synthesize novel alkylating compounds and test their in vitro ability to alkylate small methylphosphonates and methylphosphonate-peptides that mimic aged agent-AChE, to design and synthesize novel alkylating compounds specific for AChE and test their in vitro ability to alkylate aged agent-AChE and restore the ability of aged agent-AChE to be reactivated, and to down-select for the alkylating compound that will provide the most improved oxime reactivation of aged agent-AChE. The design of alkylating compounds will be guided by state-of-the-art computational methods that predict the ligand-receptor interactions of alkylating compound with aged agent-AChE, thereby providing rational guidance for the synthesis of novel therapeutics. Alkylation of model methylphosphonates, methylphosphonate-peptides and aged agent-AChE will be determined by nuclear magnetic resonance (NMR) and liquid chromatography-mass spectrometry (LC-MS), along with novel solid-phase methods. Oxime reactivation of alkylated aged agent-AChE will be determined by spectrophotometric methods.